Bacterial plant pathogens pose unique problems for disease control. One primary control strategy for bacterial diseases is based on excluding the pathogen through the use of disease free seed, or quarantine and eradication if bacterial pathogens are introduced into an area.
There are only a few chemical control agents for established bacterial diseases, and their use is often limited because of phytotoxicity or pathogen mutations resulting in resistance to the agent. Also, commonly applied protective copper compounds (for example sulfates or oxides) have limited benefit in controlling bacterial diseases because of their poor penetration into plant tissues where bacteria establish themselves and, again, mutations provide bacteria with resistance to these materials.
Unlike the control of disease outbreaks in annual crops that can be remediated in subsequent years through sanitation and the use of bacteria-free seed stocks, replanting of perennial crops such as citrus involves high capital costs to establish the planting, and several years after planting before production is initiated.
Established bacterial diseases such as those caused by Candidatus liberibacter species (citrus greening or Huanglongbing, psyllid yellows and tomato, or purple top and zebra chip of potatoes, etc.) that survive in alternate host plants in the environment and are disseminated by insect vectors that commonly infect throughout the plant life cycle are very difficult to contain because of the wide dissemination range of the insect vector and the long lag time for symptom expression (Bove, J. M. (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology 88:7-37.). Unfortunately, recent attempts to culture the organism were met with limited success (Sechler, A., Schuenzel, E. L., Cooke, P., Donnua, S., Thaveechai, N., Postnikova, E., Stone, A. L., Schneider, W. L., Damsteegt, V. D. and Schaad, N. W. (2009). Cultivation of ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ associated with Huanglongbing. Phytopathology 99, 480-486).
Huanglongbing (HLB) disease (also known as citrus greening or yellow dragon disease) is one such disease associated with the fastidious, Gram-negative, phloem-limited bacterial pathogen, Candidatus liberibacter spp. (Las). It is the most destructive citrus disease worldwide (da Graca, J. V. (1991). Citrus greening disease. Ann. Rev. Phytopathol. 29, 109-136; Halbert, S. E., and Manjunath, K. L. (2004). Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: A literature review and assessment of risk in Florida. Fla. Entomol. 87, 330-353; and Gottwald, T. R. (2010). Current epidemiological understanding of citrus Haunglongbing. Ann. Rev. Phytopathol. 48, 119-139). The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, current management practices have not been able to stop the spread of HLB disease (Duan, Y., Zhou, L., Hall, D. G., Li, W., Doddapaneni, H., Lin, H., Liu, L., Vahling, C. M., Gabriel, D. W., Williams, K. P., Dickerman, A., Sun, Y. and Gottwald, T. (2009). Complete genome sequence of citrus huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ obtained through metagenomics. Mol. Plant Microbe Interact. 22, 1011-1020).
Candidatus liberibacter species plug the plant's vascular (phloem) tissues to limit nutrient movement. Symptoms of this disease reflect a severe deficiency of essential mineral nutrients (for example copper, manganese, zinc). A temporary masking of symptoms can be achieved by applying high rates of foliar nutrients; however, the bacterial pathogen remains active and infected trees continue to decline in over-all vigor and productivity. The lag time from infection to symptom expression for this disease varies from six months to five years depending on the age of the tree, vigor, and environmental factors (Bove (2006) J Plant Pathology 88:7-37). This lag in symptom expression provides ample time for infection before detection and containment in a new area can be accomplished.
The efficacy of current strategies for management of HLB is limited and no conventional measure has shown to provide consistent and effective suppression of the disease. High cost of frequent insect control and tree removal will eventually render citrus groves unprofitable. In addition, large scale application of insecticides will disrupt the eco-system and pollute the environment (Jun, L. and Xing-Vao. J. (2005). Ecological control of forest pest: a new strategy for forest pest control. J. Forestry Res. 16, 339-342). Frequently, insecticides will become non-effective due to the acquisition of resistance. Insecticides could also kill non-target beneficial insects which disrupt the biological control currently in place.
Antibiotics injected into the tree's vascular system are often toxic to the tree, and previously available surface-applied copper compounds are not mobile enough to inhibit bacterial activity within vascular or other plant tissues. Current HLB control strategies of frequent insecticide sprays to limit populations of the psyllid insect vector, removal of infected trees, and nutrient maintenance to keep existing trees as productive as possible until they die provide little confidence for a sustainable citrus industry or incentive to reestablish it (Bove (2006) J Plant Pathology 88:7-37; UF/IFAS SWFREC, IMMOKALEE IRREC Seminar, 5 Jun. 2009).
Prokaryotes and eukaryotes have evolved numerous systems for the active export of proteins across membranes. In bacteria, the most common form of secretion of peptides with a signal sequence involves the Sec system. SecA is a protein translocase ATPase subunit that is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. It is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane (Manting, E. H., and Driessen, A. J. (2000). Escherichia coli translocase: the unravelling of a molecular machine. Mol. Microbiol. 37, 226-238). Thus, SecA is a promising antimicrobial agent because it is a protein conserved and essential in all bacteria and is absent in humans (Chen, W., Huang. Y. J., Gundala, S. R., Yang, H., Li, M., Tai, P. C. and Wang, B. (2010). The first low microM SecA inhibitors. Bioorg. Med. Chem. 18, 1617-1625; Li, M., Tai, P. C., and Wang, B. (2008). Discovery of the first SecA inhibitors using structure-based virtual screening. Biochem. Biophys. Res. Commun. 368, 839-845; and Jang, M. Y., De Jonghe, S., Segers, K., Anné, J., Herdewijn, P. (2011). Synthesis of novel 5-amino-thiazolo[4,5-d]pyrimidines as E. coli and S. aureus SecA inhibitors. Bioorg. Med. Chem. 19, 702).
SecA cooperates with the SecB chaperone to target pre-proteins to SecYEG as an active ATPase gene to drive protein translocation across the bacterial membrane when it is bound to the SecYEG complex (Economou, A., and Wickner, W. (1994). SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell. 78, 835-843). SecA is the peripheral membrane ATPase, which couples the hydrolysis of ATP to the stepwise translocation of pre-proteins (Van den Berg, B., Clemons, W. M. J., Collinson, I., Modis, Y., Hartmann, E., Harrison, S. C., and Rapoport, T. A. (2004). X-ray structure of a protein-conducting channel. Nature. 427, 36-44). The crystal structures of SecA are available for other bacteria such as Escherichia coli (Papanikolau, Y., Papadovasilaki, M., Ravelli, R. B., McCarthy, A. A., Cusack, S., Economou, A., Petratos, K. (2007). Structure of dimeric SecA, the Escherichia coli preprotein translocase motor. J. Mol. Biol. 366, 1545-1557) and the ATPase active site has been clearly defined. This structural information had been utilized for structure based design to identify antimicrobial compounds with IC50 value up to 2.5 μM against SecA of Ca. L. asiaticus (Akula, N., Zheng, H., Han, F. Q., Wang, N. (2011). Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design. Bioorg. Med. Chem. Lett. 15, 4183-4188).
Development of alternative or complementary approaches for effective management of the disease is highly desirable and will greatly help the citrus industry due to the difficulty to control the HLB disease. Considering the highly destructive nature of HLB disease and the lack of control measures, there is a huge potential to develop antimicrobial small molecules against the causal agent thus to suppress the population of Ca. L. asiaticus in plants and to reduce the inoculum for psyllid transmission. Development of antimicrobial small molecules may provide economic and ecological benefits by reducing producing costs, decreasing insecticide application, preserving the natural habitat and populations of beneficial insects, and enhancing productivity of citrus in the presence of HLB.
There is thus a need for antimicrobials that (i) are not subject to the types of antibiotic resistance currently hampering antibiotic treatment of bacteria, (ii) can be developed rapidly and with some reasonable degree of predictability as to target-bacteria specificity, (iii) are effective at low doses, meaning, in part, that they are efficiently taken up by wild-type bacteria or even bacteria that have reduced permeability for antibiotics, and (iv) show few side effects. In particular, there is currently a need in the art for an effective antimicrobial compound to target Ca. L. asiaticus. 